Liposomes, also known as phospholipid vesicles, are known to be physiologically compatible particles which provide biodegradable delivery systems for a broad range of drugs. For example, U.S. Pat. No. 5,019,369 teaches a method of targeting diagnostic and chemotherapeutic agents to tumors in the body of a patient by the intravenous administration of the agent encapsulated in unilamellar liposomes having a diameter of less than 200 nm.
Liposomes are microscopic delivery vesicles which are comprised of phospholipids which are polar molecules having a hydrophilic (ionizable) headgroup, and a hydrophobic tail consisting of fatty acid chains. Phospholipids form dosed, fluid filled spheres when properly mixed with water. The hydrophobic tails spontaneously associate and exclude the water, while the hydrophilic phosphate ester headgroups are preferentially positioned toward the water.
The result is a spherical bilayer membrane in which the fatty acid tails point towards the interior of the membrane, and the polar heads point toward the aqueous medium. The polar heads at the inner surface of the membrane point toward the liposome's aqueous interior and those at the other (outer) surface point toward the exterior aqueous medium (i.e., the external continuous phase of the liposome dispersion). Liposomes may be either multilamellar, like an onion, with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding a liquid center. Finely divided phospholipids dispersed in aqueous solution spontaneously form bilayers, and simple agitation of the mixture usually produces multilamellar vesicles (MLVs), structures having diameters of 1-10 .mu.m (1000-10,000 nm). Sonication of these structures, or other methods known in the art, leads to formation of unilamellar vesicles (UVs) having an average diameter of about 30-200 nm. The actual equilibrium diameter is largely determined by the nature of the phospholipid used, the suspending buffer, and the extent of incorporation of other lipids such as cholesterol. Standard methods for the formation of liposomes are known in the art, for example, methods for the commercial production of liposomes are described in U.S. Pat. No. 4,753,788 to Ronald C. Gamble and U.S. Pat. No. 4,935,171 to Kevin R. Bracken, the disclosures of which are incorporated herein by reference.
Liposomes for use in the invention can be prepared by any of the techniques now known in the art or subsequently developed. For example, the liposomes can be formed by the conventional technique for preparing MLVs, that is, by depositing a selected lipid on the inside wall of a suitable vesicle by dissolving the lipid in chloroform, and then evaporating the dissolvent to leave a thin film on the inside of the vessel. An aqueous solution is then added to the vessel with a swirling or vortexing motion which results in the formation of large multilamellar vesicles.
Unilamellar vesicles can be prepared by reverse phase evaporation, infusion procedures, detergent dilution and sonication, that is, by providing a shear force necessary to form the smaller, unilamellar vesicles.
In addition to liposomes, other lipid particles such as those described in U.S. Pat. No. 4,963,363 or other particles may be maintained for extended periods in a non-aggregative form, when the described anions are present on the outer surface of the particle according to the process of this invention.
It is also known to load liposomes with cationic, lipophilic drags by first forming the liposomes in an aqueous medium in the presence of an organic acid which has multiple ionizable functional groups, and exchanging the unentrapped solution for a more basic (i.e., a solution having a higher pH) solution and adding a drug to the dispersion to load the drug into the liposomes. See, for example, U.S. Pat. No. 4,946,683 to Forssen.
However, there may be potential problems with certain liposome dispersions, particularly those containing smaller liposomes or liposomes which include a multivalent anion disposed on the surface, in that vesicle aggregation or flocculation may occur upon prolonged storage in liquid form.
Accordingly, it has been a desideratum to provide a facile way to assure that aggregation of liposomes does not occur, thus extending the shell life and optimizing the production of liposomes.
The present invention provides a method for the prevention of aggregation of lipid particles (e.g., liposomes) which are dispersed in an external aqueous medium (i.e., a continuous phase) and which include a multivalent anion, the method comprising the inclusion of a divalent cation in the continuous phase. Preferably, the divalent cation is selected from the group consisting of calcium and magnesium. In terms of the relative amounts of cation and lipids which are present in the dispersion, the divalent cation is present in an mount of up to 1.5:1 mole ratio with respect to total lipid content, preferably 0.002:1 to 0.9:1, most preferably 0.002:1 to 0.6:1. For most formulations, the divalent cation may be present in the continuous phase of the liposome dispersions in an mount of up to 50 mM, preferably 0.1 to 25 mM and most preferably from 0.1 to 16 mM to provide a reliable working range. While in the examples which follow the cation is added to the liposome dispersion, in other formulations the cation might be added to the hydrating buffer solution prior to liposome formation and have a similar anti-aggregative effect if the metal ion remains present in the external phase.
The divalent cation is added to the dispersion in the form of a salt, most preferably a salt selected from the group consisting chloride, bromide, iodide, nitrate, nitrite or carbonate. Most preferably, the divalent cation is calcium and is in the form of a soluble salt.
The multivalent anion of the liposome is selected from the group consisting of citrate, succinate, tartrate, oxalate, isocitrate, glutarate, fumarate, maleate, malonate, adipate, phthalate and dextran sulfate. The multivalent anion is usually found on the liposome in residual form, e.g., adhering to the liposome following a buffer replacement. The residual multivalent anion is thought to be a principal cause of aggregation in such liposomes.